Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting

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Abstract
To investigate the spontaneous frequency of occurrence of stable multidrug‐resistant cells in a population of drug‐sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence‐activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrugresistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrugresistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr‐content sort window. Net growth of cells in culture was observed 15–20 days after sorting, indicating that of the P388/S cells collected from the low dnr‐content sort window, very few were actually highly drug‐resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug‐resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross‐resistant to vincristine and actinomycin‐D, and distinct from P388/S, possessed a 150‐ to 160–kD membrane species identified by Vinca alkaloid photoaffinity labeling. The cytogenetic alterations found in P388/LoSort were different from those found in P388/adr, confirming that P388/LoSort cells are unique, although phenotypically similar to P388/adr. These studies suggest that spontaneous mutation to a highly multidrug‐resistant phenotype does exist in P388/S cells, but that the mutation frequency is low, with an estimated frequency of 2 × 10−8.